PI-PLCh is involved in the modulation of the proximal tubule Na + -ATPase by angiotensin II L.B.A. Rangel, A.G. Lopes, L.S.M. Lara, T.L.G. Carvalho, I.V. Silva, M.M. Oliveira, M. Einicker-Lamas, A. Vieyra, L. Nogaroli, C. Caruso-Neves * Instituto de Biofı ´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,CCS Bloco G, 21949-900, Rio de Janeiro, RJ, Brazil Received 19 July 2004; accepted 1 December 2004 Available online 20 December 2004 Abstract In previous papers we showed that Ang II increases the proximal tubule Na + -ATPase activity through AT 1 /PKC pathway [L.B. Rangel, C. Caruso-Neves, L.S. Lara, A.G. Lopes, Angiotensin II stimulates renal proximal tubule Na + -ATPase activity through the activation of protein kinase C. Biochim. Biophys. Acta 1564 (2002) 310–316, L.B.A. Rangel, A.G. Lopes, L.S. Lara, C. Caruso-Neves, Angiotensin II stimulates renal proximal tubule Na + -ATPase activity through the activation of protein kinase C. Biochim. Biophys. Acta 1564 (2002) 310–316]. In the present paper, we study the involvement of PI-PLCh on the stimulatory effect of angiotensin II (Ang II) on the proximal tubule Na + -ATPase activity. Western blotting assays, using a polyclonal antibody for PI-PLCh, show a single band of about 150 KDa, which correspond to PI-PLCh isoforms. Ang II induces a rapid decrease in PIP 2 levels, a PI-PLCh substrate, being the maximal effect observed after 30 s incubation. This effect of Ang II is completely abolished by 5Â10 À8 M U73122, a specific inhibitor of PI-PLCh. In this way, the effect of 10 À8 M Ang II on the proximal tubule basolateral membrane (BLM) Na + -ATPase activity is completely abolished by 5Â10 À8 M U73122. The increase in diacylglycerol (DAG) concentration, an product of PI-PLCh, from 0.1 to 10 nM raises the Na + -ATPase activity from 6.1F0.2 to 13.1F1.8 nmol Pi mg À1 min À1 . This effect is similar and non-additive to that observed with Ang II. Furthermore, the stimulatory effect of 10 nM DAG is completely reversed by 10 À8 M calphostin C (Calph C), an inhibitor of PKC. Taken together these data indicate that Ang II stimulates the Na + -ATPase activity of proximal tubule BLM through a PI-PLCh/PKC pathway. D 2004 Elsevier B.V. All rights reserved. Keywords: Na + -ATPase; Angiotensin II; PI-PLC; Proximal tubule; Furosemide; Kidney 1. Introduction Angiotensin II (Ang II) is an important modulator of the renal sodium excretion [3,4]. This effect is partially due to the modulatory action of Ang II on the sodium transporters expressed in the proximal tubule [3–5]. The effects of Ang II on the renal proximal tubule sodium reabsorption are principally mediated by AT 1 receptors [3–6] and have been associated to the modulation of three different sodium transporters: (1) the basolateral Na + /HCO À 3 exchanger [7]; (2) the basolateral (Na + +K + )ATPase [8]; and (3) the luminal Na + /H + exchanger [9]. More recently, our group identified another sodium transporter triggered by the Ang II/AT1 receptor complex: the furosemide-sensitive, ouabain-insen- sitive basolateral membrane Na + -ATPase [1,2]. Physiological responses to Ang II result from the activation of distinct signal transduction elements [10–13]. In this regard, Ang II stimulates renal sodium reabsorption by inhibiting adenylyl cyclase/protein kinase A (PKA) pathway or activating protein kinase C (PKC) pathway [10–12]. However, many aspects of the molecular mecha- 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.12.007 Abbreviations: Ang II, angiotensin II; ATP, adenosine triphosphate; cAMP, adenosine 3V,5V-cyclic monophosphate; EDTA, ethylenediaminete- traacetic acid; GDPhS, guanosine 5V-O-(2-thiodiphosphate); HEPES, N-2-hydroxyethylpiperazine NV-2-ethanesulfonic acid; PMSF, phenylme- thylsulfonyl fluoride; Tris, tris(trishydroxymethyl)-aminomethane; PIP 2 , phosphatidylinositol-4,5-bis -phosphate; DAG, diacylglycerol; BLM, baso- lateral membrane. * Corresponding author. Tel./fax: +55 21 2280 8193. E-mail address: caruso@biof.ufrj.br (C. Caruso-Neves). Regulatory Peptides 127 (2005) 177 – 182 www.elsevier.com/locate/regpep